Kits, methods, and single-cell library preparation techniques for the dissociation of marine animal tissues

Abstract

This invention provides a kit, method, and single-cell library construction method for the dissociation of marine animal tissues. The dissociation method includes: S1) dissociating marine animal tissue samples at 8℃~12℃ using a cell dissociation buffer to obtain a single-cell suspension; S2) centrifuging the single-cell suspension and washing the single-cell precipitate obtained after centrifugation with a cell resuspension buffer to obtain single cells from the marine animal tissue sample; wherein, the cell dissociation buffer comprises a protease; the protease is Bacillus subtilis protease A; the concentration of sodium ions in the cell resuspension buffer is 300-800mM, and the concentration of potassium ions is 5-20mM.

Description

Technical Field

[0001] This invention relates to the field of single-cell sequencing of marine animal tissues, and more specifically, to a kit, method, and single-cell library preparation method for the dissociation of marine animal tissues. Background Technology

[0002] Single-cell sequencing is a core technology in current biological research, capable of detecting the genome or transcriptome of a single cell. This allows researchers to systematically study complex biological processes at a higher resolution from the single-cell level. Currently, research on marine organisms is gradually becoming one of the hottest topics in the field of biology. Single-cell sequencing brings a completely new analytical perspective to the study of marine organisms.

[0003] Single-cell sequencing requires dissociating tissue into single-cell suspensions. Obtaining high-quality single-cell sequencing data necessitates starting with single-cell suspensions with high cell viability and minimal extracellular components (such as cell debris). Currently, single-cell sequencing research on marine animals faces many challenges.

[0004] Many existing animal tissue dissociation kits are designed for human and mouse samples, but not for marine animal tissues. Existing cell dissociation kits typically operate at a reaction temperature of 37°C. However, marine animals live in seawater, where temperatures generally range from 2°C to 30°C. Using existing animal tissue dissociation kits for single-cell dissociation at high temperatures for extended periods could induce apoptosis, resulting in low-quality cell suspensions and limiting the progress of single-cell research on marine animals.

[0005] Therefore, there is a need to develop a new cell dissociation kit for marine animal tissues to overcome the problems existing in the separation of single cells from marine organisms. Summary of the Invention

[0006] The main objective of this invention is to provide a kit, method, and single-cell library construction method for the dissociation of marine animal tissues, in order to solve the problem of low cell viability of single cells obtained by dissociating marine animal tissues using existing methods.

[0007] To achieve the above objectives, according to a first aspect of the present invention, a method for dissociating marine animal tissue is provided, the method comprising: S1) dissociating a marine animal tissue sample at 8°C to 12°C using a cell dissociation buffer to obtain a single-cell suspension; S2) centrifuging the single-cell suspension and washing the single-cell precipitate obtained after centrifugation using a cell resuspension buffer to obtain single cells from the marine animal tissue sample; wherein the cell dissociation buffer comprises a protease; the protease is Bacillus subtilis protease A; the concentration of sodium ions in the cell resuspension buffer is 300-800 mM, and the concentration of potassium ions is 5-20 mM.

[0008] Furthermore, the concentration of the protease in the cell dissociation buffer was 8 mg / mL-15 mg / mL.

[0009] The cell dissociation buffer further includes EDTA; preferably, the concentration of EDTA in the cell dissociation buffer is 0.3-0.6 mM.

[0010] The cell resuspension buffer further comprises NaCl, Na2SO4, KCl, NaHCO3, Tris-Cl, EGTA, and BSA;

[0011] Preferably, the cell resuspension buffer comprises 300-600mM NaCl, 5-20mM KCl, 20-50mM Na2SO4, 1-10mM NaHCO3, 5-20mM Tris-Cl, 0.5-1mM EGTA, and 0.5%-1% BSA by volume.

[0012] Furthermore, in S1), the dissociation time is 30–60 min.

[0013] To achieve the above objectives, according to a second aspect of the present invention, a kit for the dissociation of marine animal tissue cells is provided, the kit comprising a cell dissociation buffer and a cell resuspension buffer; the cell dissociation buffer comprises a protease; the protease is Bacillus subtilis protease A; the concentration of sodium ions in the cell resuspension buffer is 300-800 mM and the concentration of potassium ions is 5-20 mM.

[0014] Furthermore, the concentration of the protease in the cell dissociation buffer is 8 mg / mL-15 mg / mL; preferably, the cell dissociation buffer also includes EDTA; preferably, the concentration of EDTA in the cell dissociation buffer is 0.3-0.6 mM.

[0015] Further, the cell resuspension buffer comprises NaCl, Na2SO4, KCl, NaHCO3, Tris-Cl, EGTA, and BSA; preferably, the cell resuspension buffer comprises 300-600mM NaCl, 5-20mM KCl, 20-50mM Na2SO4, 1-10mM NaHCO3, 5-20mM Tris-Cl, 0.5-1mM EGTA, and 0.5%-1% BSA by volume.

[0016] To achieve the above objectives, according to a third aspect of the present invention, a method for constructing a single-cell library of marine animals is provided. The method includes: dissociating a marine animal tissue sample using the above-described method for dissociating marine animal tissues or the above-described kit for dissociating marine animal tissue cells to obtain single cells of the marine animal tissue sample; resuspending the single cells using a cell resuspension buffer to obtain a single-cell resuspension; and constructing a single-cell library using the single-cell resuspension.

[0017] Furthermore, the concentration of single cells in the single-cell resuspension is 800-1200 cells / μL.

[0018] To achieve the above objectives, according to a fourth aspect of the present invention, the application of the above-described method for dissociating marine animal tissues, or the above-described kit for dissociating marine animal tissue cells, or the above-described method for constructing a single-cell library of marine animals, in the construction of a single-cell library of marine animals is provided.

[0019] By applying the technical solution of this invention, marine animal tissue samples are enzymatically digested using a cell dissociation buffer containing Bacillus subtilis protein A at a low temperature of 8℃ to 12℃ to obtain a single-cell suspension with high cell viability. The single cells are then washed with a cell resuspension buffer containing 300-800 mM sodium ions and 5-20 mM potassium ions, thus maintaining osmotic pressure balance and high cell viability in the marine animal tissue samples. This meets the requirements for subsequent single-cell library construction and lays the foundation for single-cell research on marine animals. Attached Figure Description

[0020] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0021] Figure 1 The image shows a microscopic (10x objective) examination of a single-cell suspension obtained from the preparation of gill tissue of a deep-sea tubular worm according to Example 2 of the present invention.

[0022] Figure 2 The diagram shows the peak shape of cDNA products from the gill tissue of a deep-sea tubular worm, as described in Example 2 of this specification, for the construction of a single-cell transcriptome library.

[0023] Figure 3 The diagram shows the result of library peak plots for library construction using gill tissue from deep-sea tubular worms in Example 2 of this specification.

[0024] Figure 4 The image shows a microscopic (20x objective) examination result of a single-cell suspension prepared from deep-sea coral polyps in Example 3 of this specification.

[0025] Figure 5 The image shows a microscopic examination (10x objective) of a single-cell suspension obtained from the preparation of gill tissue of a shallow-sea mollusk in Example 4 of this specification.

[0026] Figure 6 The image shows a microscopic (10x objective) examination result of a single-cell suspension obtained from the preparation of gill tissue of the striped bamboo shark, a marine fish, according to Example 5 of the present invention.

[0027] Figure 7 The image shows a microscopic (10x objective) examination result of a single-cell suspension obtained from the preparation of kidney tissue of the marine fish striped bamboo shark in Example 5 of the present invention.

[0028] Figure 8 The image shows a microscopic (10x objective) examination result of a single-cell suspension prepared from the intestinal tissue of a marine fish, the striped bamboo shark, according to Example 5 of the present invention.

[0029] Figure 9 The image shows a microscopic (10x objective) examination result of a single-cell suspension obtained from sea cucumber intestinal tissue preparation in Example 6 of the present invention.

[0030] Figure 10 The image shows a microscopic (10x objective) examination result of a single-cell suspension of gill tissue from a shallow-sea mollusk in Example 7 of this specification.

[0031] Figure 11 The image shows a microscopic (10x objective) examination result of a single-cell suspension of sea cucumber intestinal tissue in Example 8 of the present invention.

[0032] Figure 12 The image shows a microscopic (10x objective) examination result of a single-cell suspension of deep-sea coral polyp tissue in Comparative Example 1-A according to the present invention.

[0033] Figure 13The image shows a microscopic (10x objective) examination of a single-cell suspension of gill tissue from a deep-sea tubular worm in Comparative Example 2-A according to the present invention.

[0034] Figure 14 The diagram shows the peak shape of cDNA products from a single-cell transcriptional library of gill tissue from deep-sea tubular worms, as described in Comparative Example 2 of this invention.

[0035] Figure 15 The diagram shows the library peaks of a single-cell transcriptome assembly library of gill tissue from deep-sea tubular worms, as described in Comparative Example 2 of this invention.

[0036] Figure 16 The image shows a microscopic (10x objective) examination result of a single-cell suspension of sea cucumber intestinal tissue in Comparative Example 3 according to the present invention.

[0037] Figure 17 The image shows a microscopic (20x objective) examination result of a single-cell suspension of sea cucumber intestinal tissue in Comparative Example 4 according to the present invention.

[0038] Figure 18 The image shows a microscopic examination (10x objective) of a single-cell suspension of coral polyp tissue in Comparative Example 5 according to the present invention.

[0039] Figure 19 The image shows a microscopic (10x objective) examination result of a single-cell suspension of shellfish gill tissue in Comparative Example 6 according to the present invention. Detailed Implementation

[0040] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0041] As mentioned in the background section, existing methods for isolating single cells from marine animals suffer from low cell viability. This is because the living environment of marine organisms differs from laboratory conditions. Marine environmental temperatures vary between 2°C and 30°C, while existing animal dissociation kits typically recommend a temperature of 37°C. Marine animal cells are prone to stress during the dissociation process at 37°C, leading to a large number of cell deaths and a low cell survival rate.

[0042] Therefore, in this application, the inventors attempt to develop a new method for the dissociation of marine animal tissues. The cell dissociation buffer used in the dissociation process can still fully dissociate marine animal tissue samples under simulated ocean temperature conditions. The cell resuspension buffer used for washing and suspending single cells of marine animal tissue samples provides suitable osmotic pressure conditions for marine animal single cells, which is beneficial to improving the overall separation effect of single cells of marine animal tissues.

[0043] In a first typical embodiment of this application, a method for dissociating marine animal tissue is provided. The method includes: S1) dissociating a marine animal tissue sample using a cell dissociation buffer at 8°C to 12°C (including but not limited to 8°C, 9°C, 10°C, 11°C, or 12°C) to obtain a single-cell suspension; S2) centrifuging the single-cell suspension and washing the resulting single-cell precipitate with a cell resuspension buffer to obtain single cells from the marine animal tissue sample; wherein the cell dissociation buffer comprises... Including proteases; the protease is Bacillus subtilis protease A (CAS: 9014-01-1). The sodium ion concentration in the cell resuspension buffer is 300-800 mM (including but not limited to 300, 350, 400, 450, 500, 550, 600, 650, 700, 750 or 800 mM), and the potassium ion concentration is 5-20 mM (including but not limited to 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 mM).

[0044] In existing animal tissue dissociation kits, collagenase I or trypsin is commonly used to enzymatically digest animal tissue samples to obtain single cells. However, the recommended temperature for these kits is generally 37°C. Marine animal cells are prone to stress during dissociation at 37°C, leading to significant cell death and low cell survival rates. Furthermore, most enzymes used for dissociating animal tissues, including but not limited to collagenase I, trypsin, and neutral protease II, struggle to maintain high catalytic activity at low temperatures, making it difficult to dissociate marine animal tissues and obtain high-quality single cells.

[0045] The cell dissociation buffer of this application contains Bacillus subtilis protein A, which enables efficient (within 1 hour) dissociation of single-cell suspensions of marine animal tissues at low temperatures (8℃-12℃). Furthermore, the single-cell suspension exhibits minimal cell aggregation, maintains a high cell viability (greater than 80%), and contains minimal cell debris and aggregates. This demonstrates that Bacillus subtilis protein A maintains high activity even at low temperatures, ensuring thorough dissociation of marine animal tissue samples, preventing alterations in gene expression of marine animal cells, improving the viability of single cells, and enhancing the overall quality of the single-cell suspension.

[0046] This application controls the sodium and potassium ion concentrations of the cell resuspension buffer within the aforementioned range. During the washing process, it can simulate a seawater environment while providing suitable osmotic pressure conditions for single cells of marine animals. This allows the single cells of marine animals to maintain a relatively intact morphology and a high viability, without interfering with subsequent downstream single-cell library construction reactions, which is beneficial to improving the quality of single-cell transcriptome sequencing data.

[0047] The method for dissociating marine animal tissues presented in this application can efficiently obtain highly active and high-yield marine animal single-cell suspensions. In a specific embodiment of this application, existing single-cell library construction techniques were used for marine animal single-cell transcriptome library construction and sequencing, resulting in high-quality data analysis results. This further verifies the high cell viability and high quality of the marine animal single-cell suspensions obtained in this application. The marine animal single cells obtained using the method for dissociating marine animal tissues presented in this application are applicable to various scientific research fields such as single-cell genomics and single-cell transcriptomics, contributing to the advancement of marine animal research and laying an important research foundation for in-depth exploration of the environmental adaptation mechanisms of marine animals.

[0048] In a preferred embodiment, the concentration of the protease in the cell dissociation buffer is 8 mg / mL-15 mg / mL; preferably, the cell dissociation buffer further comprises EDTA; preferably, the concentration of EDTA in the cell dissociation buffer is 0.3-0.6 mM. Preferably, the cell dissociation buffer further comprises calcium- and magnesium-free DPBS for dissolving the protease powder.

[0049] By controlling the concentration ratio of each component in the cell dissociation process in this application within the above-mentioned range, the dissociation effect of marine animal tissue samples can be promoted, and the cell viability and single cell quality in the cell suspension can be improved.

[0050] In a preferred embodiment, the cell resuspension buffer comprises NaCl, Na2SO4, KCl, NaHCO3, Tris-Cl, EGTA, and BSA; preferably, the cell resuspension buffer comprises 300-600 mM NaCl, 5-20 mM KCl, 20-50 mM Na2SO4, 1-10 mM NaHCO3, 5-20 mM Tris-Cl, 0.5-1 mM EGTA, and 0.5%-1% BSA by volume.

[0051] By controlling the concentration ratio of each component in the cell resuspension buffer in this application within the above-mentioned range, it not only simulates the complex chemical system of seawater, but also can be used in conjunction with the low-temperature tissue dissociation buffer of this application to maintain the concentration of sodium and potassium ions. This is beneficial to maintaining the osmotic pressure of marine animal single cells, improving cell viability, avoiding interference with downstream single-cell library construction, and promoting the successful construction of marine biological single-cell libraries.

[0052] In a preferred embodiment, in S1), the dissociation time is 30 to 60 minutes, including but not limited to 30, 40, 50 or 60 minutes.

[0053] When using the cell dissociation buffer of this application to dissociate marine animal tissues, the tissue enzymatic digestion is observed under a microscope. Depending on the total number of cells obtained after dissociation, the tissue enzymatic digestion time can be appropriately extended, but not exceeding 60 minutes.

[0054] In a second typical embodiment of this application, a kit for the dissociation of marine animal tissue cells is provided. The kit includes a cell dissociation buffer and a cell resuspension buffer. The cell dissociation buffer is composed of a protease. The protease is derived from Bacillus licheniformis. The concentration of sodium ions in the cell resuspension buffer is 300-800 mM, and the concentration of potassium ions is 5-20 mM.

[0055] The kit for dissociating marine animal tissues and cells described in this application enables the dissociation of various marine animal tissue samples, yielding single-cell suspensions with high cell viability and high-quality single cells. These cells can be successfully applied to the subsequent construction of high-quality single-cell libraries. This kit is applicable to various scientific research fields, including marine animal single-cell genomics and single-cell transcriptomics, laying an important research foundation for in-depth exploration of the environmental adaptation mechanisms of marine animals and promoting the development of marine animal research.

[0056] In a preferred embodiment, the concentration of the protease in the cell dissociation buffer is 8 mg / mL-15 mg / mL; preferably, the cell dissociation buffer further comprises EDTA; preferably, the concentration of EDTA in the cell dissociation buffer is 0.3-0.6 mM; preferably, the cell resuspension buffer comprises NaCl, Na2SO4, KCl, NaHCO3, Tris-Cl, EGTA, and BSA; preferably, the cell resuspension buffer comprises 300-600 mM NaCl, 5-20 mM KCl, 20-50 mM Na2SO4, 1-10 mM NaHCO3, 5-20 mM Tris-Cl, 0.5-1 mM EGTA, and 0.5%-1% BSA by volume.

[0057] In a third typical embodiment of this application, a method for constructing a single-cell library of marine animals is provided. The method includes: dissociating a marine animal tissue sample using the above-mentioned method for dissociating marine animal tissues or the above-mentioned kit for dissociating marine animal tissue cells to obtain single cells of the marine animal tissue sample; resuspending the single cells using a cell resuspension buffer to obtain a single-cell resuspension; and constructing the single-cell library using the single-cell resuspension.

[0058] In existing microfluidic single-cell library construction systems, single cells are typically suspended in PBS + 0.04% BSA for droplet generation. However, marine animal single cells suspended in PBS + 0.04% BSA cannot maintain the osmotic pressure environment, leading to cell death and fragmentation. This results in intracellular mRNA being released outside the cell, causing poor quality single-cell sequencing data. Therefore, there is a need to develop a marine animal cell resuspension buffer that can be used for cell washing during tissue dissociation and for suspending single cells during single-cell library construction. This buffer should maintain cell viability without interfering with downstream single-cell library construction reactions, providing suitable osmotic pressure conditions for marine animal single cells, allowing them to maintain a relatively intact morphology during the water-in-oil droplet generation process.

[0059] The cell resuspension buffer described in this application can maintain the viability of single cells during the washing process of cell dissociation. It is also compatible with existing single-cell transcriptome library assembly systems based on droplet microfluidics, ensuring the normal operation of the water-in-oil system and providing suitable osmotic pressure conditions for marine single cells, allowing them to maintain a relatively intact morphology when entering the water-in-oil emulsion. Using single-cell transcriptome library assembly enables the differentiation of different cells and transcripts through sequence tags, obtaining high-quality sequencing data and significantly improving the capture efficiency of marine cells.

[0060] In a preferred embodiment, the concentration of single cells in the single-cell resuspension is 800-1200 cells / μL, including but not limited to 800, 850, 900, 950, 1000, 1100 or 1200 cells / μL.

[0061] In a fourth typical embodiment of this application, the above-described method for dissociating marine animal tissues, or the above-described kit for dissociating marine animal tissue cells, or the above-described method for constructing a single-cell library of marine animals, is provided for use in constructing a single-cell library of marine animals.

[0062] The beneficial effects of this application will be explained in more detail below with reference to specific embodiments.

[0063] Unless otherwise specified, the reagents used in the embodiments of this application are all commercially available products.

[0064] Example 1

[0065] Information on the raw materials and instruments used in this embodiment is shown in Table 1.

[0066] Table 1

[0067] Raw materials / instruments factory Bacillus subtilis protease A(1) Sigma part number P5380 Bacillus subtilis protease A(2) Megazyme product code E-BSPRPD Bacillus subtilis protease A(3) Perfemiker part number PN01121 Collagenase I(1) Gibco product number 17100017 Collagenase I(2) Invitrogen product number 17100-017 Trypsin(1) Gibco product number 25200072 Trypsin(2) HyClone part number SH30042.01 Neutral protease (Dispase II) (1) Gibco product number 17105041 Neutral protease (Dispase II)(2) Yeasen product number 40104ES80 Calcium and magnesium ion-free DPBS Invitrogen 0.5M EDTA Invitrogen Bovine serum albumin (BSA) powder Sigma Surgical blade Golden Ring Medical 30μm cell sieve Miltenyi Biotec 1mL wide-mouth pipette tip AXYGEN 15mL conical centrifuge tube BD 10cm cell culture dish BD microscope ZEISS Horizontal rotor refrigerated centrifuge Eppendorf Vertical Mixer HulaMixer Thermo

[0068] Preparation and usage of kits for the dissociation of marine animal tissues and cells:

[0069] (1) Preparation of the reagent kit

[0070] The kit consists of the following components: cell dissociation buffer and cell resuspension buffer.

[0071] The cell dissociation buffer is a dissociation reagent with a total volume of 2 mL. The specific components are shown in Table 2. Prepare the cell dissociation buffer according to the components and volumes shown in Table 2.

[0072] Table 2

[0073] Components volume 100 mg / mL Bacillus subtilis proteinase A(1) 200μL 0.5M EDTA 2μL Calcium and magnesium ion-free DPBS 1798μL

[0074] The marine cell resuspension buffer is a cell washing reagent with a total volume of 100 mL. Its specific components are shown in Table 3. Take each component according to Table 3, dissolve and mix thoroughly, filter using a 0.22 μm filter membrane, and collect the filtrate, which is the marine cell resuspension buffer.

[0075] Table 3

[0076] Components volume 5M NaCl 9mL <![CDATA[Sodium sulfate powder]]> 0.47g 1M KCl 0.9mL <![CDATA[Sodium bicarbonate powder]]> 0.02g 1M Tris-Cl pH 8.2 1mL 0.2M EGTA 1.2mL Bovine serum albumin (BSA) powder 0.5g Nuclease-free water Add water to 50mL

[0077] (2) How to use the kit

[0078] 1) Tissue Dissection: Freshly collected marine animals should be dissected immediately. Take 0.5-1g of the target tissue (e.g., fish gill tissue, coral polyp tissue) and place it in a 10cm culture dish. Wash the tissue with 1mL of pre-cooled DPBS. Then, use sterile scissors or a scalpel to cut the tissue into pieces approximately 5mm in size. Tissue disruption must be performed on an ice pack, and the processing time should be controlled within 5 minutes; otherwise, RNA degradation may occur.

[0079] 2) Tissue digestion: Using a 1 mL wide-mouth pipette tip, transfer the chopped tissue into cell dissociation buffer (approximately 2 mL of cell dissociation buffer in a 5 mL centrifuge tube). Incubate at 8-12°C for 30 min, mixing continuously using a vertical mixer during the process.

[0080] 3) Cell washing: Filter the single-cell suspension obtained in the previous step through a 30μm cell sieve, and wash off any remaining cells on the sieve with 2mL of marine cell resuspension buffer to ensure complete cell collection. Transfer the collected cells to a 15mL centrifuge tube and centrifuge at 300g for 5 minutes at 4°C to collect the cell pellet. Wash the cells twice with 8mL of marine cell resuspension buffer. Centrifuge at 300g for 5 minutes at 4°C and discard the supernatant.

[0081] 4) Cell microscopy: Resuspend cells in 500 μL of cell resuspension buffer. Using a cell counting chamber under a microscope, determine cell viability and number according to the trypan blue staining method. Normal living cells have intact cell membranes that repel trypan blue and remain unstained; however, dead cells have incomplete cell membranes with increased permeability, allowing trypan blue to penetrate and stain the cells blue.

[0082] Cell viability is calculated by the ratio of surviving cells to the total number of cells, as observed and counted under a microscope.

[0083] 5) Library construction and sequencing: The cell concentration was adjusted to 800-1200 cells / μL using marine cell resuspension buffer. Library construction was performed using a single-cell transcription library construction and sequencing platform (DNBelab C4 platform) according to standard procedures, followed by sequencing and analysis (Agilent 2100).

[0084] The kit for dissociating marine animal tissues and cells of this application was applied to dissociate different marine animal tissue samples, as shown in Examples 2 to 6.

[0085] Example 2

[0086] The sample was gill tissue of a deep-sea tubular worm. The tissue was dissociated according to the steps in Example 1 to obtain a single-cell library.

[0087] The microscopic examination results (10x objective) of the single-cell suspension prepared in this embodiment are as follows: Figure 1 As shown. In this embodiment, the cell viability was 85%, the cells were round with intact boundaries and no cell debris.

[0088] The peak shape of the cDNA product constructed from the single-cell transcriptome library in this embodiment is as follows: Figure 2 As shown in the figure, the main peak of cDNA is around 1000bp, and the size of the cDNA fragment is within the normal range, indicating that the RNA integrity is high during cell dissociation.

[0089] The results of the library peak plots constructed from the single-cell transcriptome library in this embodiment are as follows: Figure 3 As shown. By Figure 3 As can be seen, the size of the single-cell library in this embodiment is in the range of 300bp-600bp, which is within the normal range. After sequencing, the data were subjected to quality control analysis, and the sequencing analysis results are shown in Table 4.

[0090] Table 4

[0091] Analysis indicators data Cell number estimation 14,369 Average number of reads per cell 36,209 Average number of UMIs per cell 2,065 Total number of genes detected 16,255 Average number of genes per cell 509

[0092] The results showed that the single-cell suspension obtained using this kit met the requirements for single-cell sequencing and yielded good sequencing results.

[0093] Example 3

[0094] The sample was deep-sea coral polyp tissue, and the tissue was dissociated according to the steps in Example 1. The protease was Bacillus subtilis protease A (2).

[0095] The microscopic examination results (20x objective) of the single-cell suspension in this embodiment are as follows: Figure 4 As shown, the cell viability was 95%, with no cell debris or cell aggregation.

[0096] Example 4

[0097] The sample was gill tissue of a shallow-sea shellfish. The tissue was dissociated according to the steps in Example 1. The protease was Bacillus subtilis protease A (3).

[0098] The microscopic examination results (10x objective) of the single-cell suspension in this embodiment are as follows: Figure 5 As shown, the cell viability was 87%, the cell morphology was intact, and there were no cell fragments or cell clusters.

[0099] Example 5

[0100] The samples were gills, kidneys, and intestinal tissues of the striped bamboo shark, a marine fish. The tissues were dissociated according to the steps in Example 1. The proteases were Bacillus subtilis protease A (1), Bacillus subtilis protease A (2), and Bacillus subtilis protease A (3).

[0101] The microscopic examination results (10x objective) of the single-cell suspension obtained from the gill tissue of the striped bamboo shark in this embodiment are as follows: Figure 6 As shown, the cell viability was 87%, with little cell debris and cell aggregation.

[0102] The microscopic examination results (10x objective) of the single-cell suspension obtained from the kidney tissue of the marine fish striped bamboo shark in this embodiment are as follows: Figure 7 As shown, the cell viability was 95%, with little cell debris and cell aggregation.

[0103] In this embodiment, the microscopic examination results (10x objective) of the single-cell suspension prepared from the intestinal tissue of the marine fish striped bamboo shark are as follows: Figure 8 As shown, the cell viability was 90%, with little cell debris and cell aggregation.

[0104] Example 6

[0105] The sample was sea cucumber intestinal tissue, and the tissue was dissected according to the steps in Example 1.

[0106] The microscopic examination results (10x objective) of the single-cell suspension in this embodiment are as follows: Figure 9 As shown, the cell viability was 91%, with little cell debris and cell aggregation.

[0107] Example 7

[0108] Using gill tissue from shallow-sea shellfish as a sample, the only difference from Example 1 was that the final concentration of protease in the cell dissociation buffer was 20 mg / mL and the final concentration of EDTA was 1 mM. The remaining steps were the same as in Example 1.

[0109] The microscopic examination results of the single-cell suspension microscope (10x objective) in this embodiment are as follows: Figure 10 As shown in the figure, the cell viability was 50%, the total number of cells obtained after dissociation was small, and some cells were broken.

[0110] Example 8

[0111] Using sea cucumber intestinal tissue as a sample, the only difference from Example 1 is that the final concentration of NaCl in the cell resuspension buffer in this example is 100mM, the final concentration of KCl is 50mM, and the final concentrations of other reagent components are the same as those in the cell resuspension buffer in Example 1.

[0112] The microscopic examination results of the single-cell suspension microscope (10x objective) in this embodiment are as follows: Figure 11 As shown, the viability of the cells obtained after dissociation is about 60%, and the cells exhibit clustering, with a small proportion of single intact cells.

[0113] Comparative Example 1

[0114] A: Using deep-sea coral polyp tissue as a sample, the only difference from Example 1 is that the protease in Example 1 is replaced with collagenase I (1) at a final concentration of 2 mg / mL in the cell dissociation buffer, and the dissociation temperature in step 2 is 37°C.

[0115] The microscopic examination results of the single-cell suspension in this comparative example (10x objective lens) are as follows: Figure 12 As shown, the total number of cells obtained after dissociation was small, the cell viability was 40%, and the content of cell debris and other impurities was relatively high.

[0116] B: Using deep-sea coral polyp tissue as a sample, the only difference from Example 1 was that the protease in Example 1 was replaced with collagenase I at a final concentration of 2 mg / mL in the cell dissociation buffer (2). The total number of cells obtained after dissociation was small, with more cell clusters and many undispersed cells.

[0117] Comparative Example 1 shows that during the enzyme screening process in the cell dissociation buffer of this application, replacing the protease with collagenase I, which is commonly used in the dissociation of conventional animal samples, resulted in poor dissociation efficiency and low cell viability. Furthermore, collagenase I, commonly used in animal tissue dissociation, is difficult to activate at low temperatures. In conclusion, low-temperature dissociation conditions play a crucial role in the successful dissociation of marine animal cells, and commonly used low-temperature tissue dissociation proteases are difficult to activate under low-temperature conditions.

[0118] Comparative Example 2

[0119] A: Using deep-sea tubular worm gill tissue as a sample, the only difference from Example 1 was that the protease in Example 1 was replaced with trypsin (1) at a volume percentage of 0.25% in the cell dissociation buffer. In step 2, the dissociation temperature was 37°C.

[0120] The marine cell resuspension buffer used in this comparative example was PBS buffer containing 0.04% BSA.

[0121] The microscopic examination results of the single-cell suspension in this comparative example (10x objective lens) are as follows: Figure 13 As shown, the viability of the dissociated cells was about 60%, with a relatively high content of impurities such as rod cell fragments.

[0122] Library construction was performed using the single-cell transcription library construction sequencing platform, following step 5 of Example 1.

[0123] The peak shape results of the cDNA product from the single-cell transcription library in this comparative example are as follows: Figure 14 As shown in the figure, the main peak of cDNA is around 500bp, and the cDNA fragment size is relatively small, suggesting that RNA may have been degraded during cell dissociation or library construction.

[0124] The peak plots of the library for this comparative single-cell transcriptional assembly are as follows: Figure 15 As shown in Table 5, the library size ranged from 300bp to 600bp, and the fragment size was within the normal range. After sequencing, the data were subjected to quality control analysis, and the results are shown in Table 5.

[0125] Table 5

[0126] Analysis indicators data Cell number estimation 2,491 Average number of reads per cell 38,837 Average number of UMIs per cell 2,005 Total number of genes detected 13,738 Average number of genes per cell 402

[0127] Based on the sequencing results in Tables 4 and 5, the combination of using trypsin instead of the cell dissociation buffer of this invention and using PBS buffer containing 0.04% BSA instead of the marine cell resuspension buffer of this invention to resuspend cells resulted in a decrease in cell viability during cell dissociation (cell viability decreased from 85% to 60%). Single-cell transcriptome library sequencing results showed a significant decrease in cell capture efficiency (the number of cells captured decreased from 14,369 to 2,491 cells), and a reduction in the number of genes detected (the total number of genes detected decreased from 16,255 to 13,738).

[0128] B: Using deep-sea tubular worm gill tissue as a sample, the only difference from Example 1 was that the protease in Example 1 was replaced with trypsin at a volume percentage of 0.25% in the cell dissociation buffer (2). The total number of cells obtained after dissociation was small, with more cell clusters and many undispersed cells.

[0129] The results showed that during the screening of enzymes in the cell dissociation buffer of this application, replacing the protease with trypsin, which is commonly used in the dissociation of conventional animal samples, resulted in poor dissociation efficiency, low cell viability, and trypsin's inability to exert its activity at low temperatures. In conclusion, low-temperature dissociation conditions play a crucial role in the successful dissociation of marine animal cells, and commonly used low-temperature tissue dissociation proteases are unlikely to exert their activity under low-temperature conditions.

[0130] Comparative Example 3

[0131] The sample was sea cucumber intestinal tissue. The only difference from Example 1 was that the protease in Example 1 was replaced with a neutral protease (Dispase II) at a volume ratio of 0.1% in the cell dissociation buffer (1). The remaining steps were the same as in Example 1.

[0132] The microscopic examination results of the single-cell suspension in this comparative example (10x objective lens) are as follows: Figure 16 As shown, the total number of cells obtained after dissociation was small, the cell viability was 50%, there were many cell clusters, and many undispersed cells were present.

[0133] Comparative Example 3 showed that during the screening of enzymes in the cell dissociation buffer in this application, after replacing the protease with the neutral protease (Dispase) commonly used in the dissociation process of conventional animal samples (1), the dissociation effect of the neutral protease was poor in the dissociation environment of 8-12℃.

[0134] Comparative Example 4

[0135] The sample was sea cucumber intestinal tissue. The only difference from Example 1 was the cell dissociation buffer, in which the protease in Example 1 was replaced with a neutral protease (Dispase) at a volume percentage of 0.1% (2). In step 2, the dissociation temperature was 37°C. The remaining steps were the same as in Example 1.

[0136] The microscopic examination results of the single-cell suspension in this comparative example (20x objective lens) are as follows: Figure 17 As shown, the viability of the cells obtained after dissociation was 20%, and most of the cells had incomplete cell membranes and had died.

[0137] Comparative Example 4 showed that during the screening of enzymes in the cell dissociation buffer in this application, after replacing the protease with the neutral protease (Dispase) (2) commonly used in the dissociation process of conventional animal samples, and changing the dissociation temperature to the optimal reaction temperature of neutral protease 37℃, a large number of sea cucumber intestinal cells were broken and died, resulting in poor dissociation effect.

[0138] Comparative Example 5

[0139] The sample was deep-sea coral polyp tissue, and the only difference from Example 1 was that the dissociation temperature in step 2 was 25°C.

[0140] The microscopic examination results of the single-cell suspension in this comparative example (10x objective lens) are as follows: Figure 18 As shown, the viability of the dissociated cells was 56%, and the cell suspension contained a large amount of cell debris and other impurities.

[0141] Comparative Example 5 shows that after changing the dissociation temperature to 25℃ in the cell dissociation buffer in this application, the cell viability of coral hydroid cells decreased, and the cell suspension contained more cell debris and other impurities, resulting in poor dissociation effect.

[0142] Comparative Example 6

[0143] The sample was shellfish gill tissue, and the only difference from Example 1 was that the cell resuspension buffer was composed of PBS buffer containing 0.04% BSA.

[0144] The microscopic examination results of the single-cell suspension in this comparative example (10x objective lens) are as follows: Figure 19 As shown, the viability of the dissociated cells was 65%, and the cell suspension contained a large amount of cell debris and other impurities.

[0145] Comparative Example 5 showed that after the cell resuspension buffer in this application was replaced with PBS buffer containing 0.04% BSA, the cell viability of shellfish gill cells decreased and the dissociation effect was poor.

[0146] As can be seen from the above description, the above embodiments of the present invention achieve the following technical effects: In the method for dissociating marine animal tissues in this application, marine animal tissue samples can be enzymatically digested using a cell dissociation buffer containing a protease derived from Bacillus licheniformis at a low temperature of 8℃~12℃, and then the single-cell suspension can be washed with a cell resuspension buffer with a sodium ion concentration of 300-800mM and a potassium ion concentration of 5-20mM, so that the osmotic pressure of the single cells in the marine animal tissue sample is balanced, the cell viability is improved, and the needs of subsequent single-cell library construction can be met;

[0147] Furthermore, the aforementioned cell resuspension is compatible with existing single-cell transcriptome library construction systems based on droplet microfluidics, maintaining the normal operation of the water-in-oil system and providing suitable osmotic pressure conditions for marine single cells, allowing them to maintain a relatively intact morphology upon entering the water-in-oil emulsion. Using single-cell transcriptome library construction enables the differentiation of different cells and transcripts through sequence tags, obtaining high-quality sequencing data and significantly improving the capture efficiency of marine cells. The marine tissue dissociation method, kit, and library construction method presented in this application can be applied to various scientific research fields such as marine animal single-cell genomics and single-cell transcriptomics, laying an important research foundation for in-depth exploration of the environmental adaptation mechanisms of marine animals and promoting the development of marine animal research.

[0148] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for dissociating marine animal tissues, characterized in that, The dissociation method includes: S1) At 8℃~12℃, marine animal tissue samples were dissociated using cell dissociation buffer to obtain single-cell suspensions; S2) Centrifuge the single-cell suspension, and wash the single-cell precipitate obtained after centrifugation with cell resuspension buffer to obtain single cells from the marine animal tissue sample; The cell dissociation buffer contains components including proteases. The protease is Bacillus subtilis protease A; The concentration of sodium ions in the cell resuspension buffer is 300-800 mM, and the concentration of potassium ions is 5-20 mM.

2. The dissociation method of claim 1, wherein, The concentration of the protease in the cell dissociation buffer is 8 mg / mL to 15 mg / mL.

3. The dissociation method of claim 1, wherein, The cell dissociation buffer also includes EDTA; Preferably, the concentration of EDTA in the cell dissociation buffer is 0.3-0.6 mM.

4. The dissociation method according to claim 1, characterized in that, The cell resuspension buffer comprises NaCl, Na2SO4, KCl, NaHCO3, Tris-Cl, EGTA, and BSA. Preferably, the cell resuspension buffer comprises 300-600mM NaCl, 5-20mM KCl, 20-50mM Na2SO4, 1-10mM NaHCO3, 5-20mM Tris-Cl, 0.5-1mM EGTA, and 0.5%-1% BSA by volume.

5. The dissociation method according to claim 1, characterized in that, In S1), the dissociation time is 30 to 60 minutes.

6. A kit for the dissociation of marine animal tissue cells, characterized in that, The kit contains cell dissociation buffer and cell resuspension buffer; The cell dissociation buffer contains proteases. The protease is Bacillus subtilis protease A; the concentration of sodium ions in the cell resuspension buffer is 300-800 mM, and the concentration of potassium ions is 5-20 mM.

7. The reagent kit according to claim 6, characterized in that, The concentration of the protease in the cell dissociation buffer is 8 mg / mL-15 mg / mL; Preferably, the cell dissociation buffer further comprises EDTA; Preferably, the concentration of EDTA in the cell dissociation buffer is 0.3-0.6 mM.

8. The reagent kit according to claim 6, characterized in that, The cell resuspension buffer comprises NaCl, Na2SO4, KCl, NaHCO3, Tris-Cl, EGTA, and BSA. Preferably, the cell resuspension buffer comprises 300-600mM NaCl, 5-20mM KCl, 20-50mM Na2SO4, 1-10mM NaHCO3, 5-20mM Tris-Cl, 0.5-1mM EGTA, and 0.5%-1% BSA by volume.

9. A method for constructing a single-cell library of marine animals, characterized in that, The construction method includes: using the marine animal tissue dissociation method of any one of claims 1 to 5, or the kit for marine animal tissue cell dissociation of any one of claims 6 to 8, to dissociate the marine animal tissue sample and obtain single cells of the marine animal tissue sample; The single cells were resuspended using the cell resuspension buffer to obtain a single-cell resuspension. The single-cell library was constructed using the single-cell resuspension.

10. The construction method according to claim 9, characterized in that, The concentration of single cells in the single-cell resuspension is 800-1200 cells / μL.

11. The application of the method for dissociating marine animal tissues according to any one of claims 1 to 5, the kit for dissociating marine animal tissue cells according to any one of claims 6 to 8, or the method for constructing a single-cell library of marine animals according to any one of claims 9 or 10 in constructing a single-cell library of marine animals.

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